Studies of this laboratory have demonstrated that clinical isolates of Pseudomonas aeruginosa are subject to rapid intracellular killing by PMN leukocytes with a selective deficiency of hydrogen peroxide (H2O2). The bactericidal mechanism appears to be independent of participation of products of oxygen metabolism, required for efficient destruction of Staphylococcus aureus and many gram-negative enteric bacteria. The latter group of bacteria survive and multiply within H2O2-deficient leukocytes and appear to be relatively resistant to the proposed oxygen-independent mechanism. Comparative studies of the capacity of aerobic and anaerobic PMN leukocytes to kill Pseudomonas aeruginosa should confirm the intracellular function, independent of oxidative metabolism, of the bactericidal mechanism. A method for separation of proteins extracted from cytoplasmic granules of PMN leukocytes in one step has been developed. Studies of our laboratory have shown that Pseudomonas aeruginosa is extremely sensitive to killing by a single protein of granule extracts. We will characterize the bactericidal protein and assess its activity under physiologic conditions. Select bacterial strains susceptible and resistant to the bactericidal protein will be compared with the spectrum of microorganisms sensitive to H2O2-dependent killing. Antibody to the purified protein will be prepared and employed to confirm the granule location of the protein by direct immunofluorescence. The antibody will also be utilized in radial immunodiffusion assay of the concentration of bactericidal protein in resting PMN leukocytes and the fraction transfered to phagolysosomes during phagocytosis. These studies should make possible a greater understanding of the contribution of granule proteins, active in oxygen-independent bactericidal reactions, to the overall bactericidal capacity of intact human PMN leukocytes.